Modulator Effect of AT1 Receptor Knockdown on THP-1 Macrophage Proinflammatory Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although the findings that Angiotensin II receptor type 1 (AT1R) gene silencing is an alternative to modulate the production of proinflammatory cytokines such has TNF-α and IL-1β via NF-κB in macrophages, besides have high blood pressure decreased are interesting, numbers of points need clarifying and certain statements require further justification. These are given below.

 

<Point>

1        The authors entitled, “Modulator effect of AT1 receptor knockdown in macrophage proinflammatory activity” (lines 3-4). However, the authors used a human monocyte line THP-1 (Tsuchiya, S. et al. Int. J. Cancer 26, 171-176, 1980). Although the results are true in THP-1 cells, it is uncertain the results are true in macrophages. The authors should test other macrophages (cell lines) than THP-1 cells such as human HL-60, human MABS-1, human MOBS-1, mouseJ774A.1, mouse RAW264.7, and mouse BMA3.1A7. Alternatively, the authors change the title to “Modulator effect of AT1 receptor knockdown in THP-1 macrophage proinflammatory activity”.

2        “Caledon Laboratories, Canada” (lines 93-94) should be changed to “Caledon Laboratories, Chalk River, Canada”.

3        “Na2HPO4” (line 96) should be changed to “Na₂HPO₄”.

4        “KH2PO4” (line 96) should be changed to “KH₂PO₄”.

5        “Sigma-Aldrich, EE. UU.” (lines 96-97) should be changed to “Sigma-Aldrich, Burlington, MA”.

6        “25 cm2 flasks” (line 101) should be changed to “25 cm² flasks”.

7        “5% CO2” (line 102) should be changed to “5% CO₂”.

8        “3 x 105 cells” (line 104) should be changed to “3 x 10⁵ cells”.

9        “2 x105 cells” (line 108) should be changed to “2 x10⁵ cells”.

10     “InvivoGen” (line 114) should be changed to “Invivogen”.

11     “2 x105 cells” (line 119) should be changed to “2 x10⁵ cells”.

12     In Table 1, “NF-KB” should be changed to “NF-κB”.

13     “R&DSystems, Minneapolis, MN, USA” (lines 145-146) should be changed to “R&DSystems, Minneapolis, MN”.

14     “Bio-Rad, Calif, USA” (line 148) should be changed to “Bio-Rad, Hercules, CA”.

15     In Ref. 12, “Wang, C. H., Li, S. H., Weisel, R. D., Fedak, P. W., Dumont, A. S., Szmitko, P., ... & Verma, S.” should be changed to “Wang, C.H., Li, S.H., Weisel, R.D., Fedak, P.W., Dumont, A.S., Szmitko, P., Li, R.K., Mickle, D.A., Verma, S.”.

16     In Ref. 13, “Recent Patents on Cardiovascular Drug Discovery (Discontinued), 2(1), 23-27” should be changed to “Recent Pat Cardiovasc Drug Discov. 2007;2(1):23-27.”.

17     In Ref. 28, “Forrester, S. J., Booz, G. W., Sigmund, C. D., Coffman, T. M., Kawai, T., Rizzo, V., ... & Eguchi, S.” should be changed to “Forrester, S.J., Booz, G.W., Sigmund, C.D., Coffman, T.M., Kawai, T., Rizzo, V., Scalia, R., Eguchi, S.”.

18     In Ref. 39, “Aki, K., Shimizu, A., Masuda, Y., Kuwahara, N., Arai, T., Ishikawa, A., ... & Fukuda, Y.” should be changed to “Aki, K., Shimizu, A., Masuda, Y., Kuwahara, N., Arai, T., Ishikawa, A., Fujita, E., Mii, A., Natori, Y., Fukunaga, Y., Fukuda, Y.”.

19     In Ref. 40, “Journal of Diabetes Research, 2012” should be changed to “Exp. Diabetes Res. 2012;2012:618923.”.

20     In Ref. 49, “Valsartan, independientemente del receptor AT1 o PPAR, suprime la activación de macrófagos inducida por LPS y mejora la resistencia a la insulina en adipocitos cocultivados.” should be changed to “Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes.”.

 

 

Author Response

Reviewer 1

1. The authors entitled, “Modulator effect of AT1 receptor knockdown in macrophage proinflammatory activity” (lines 3-4). However, the authors used a human monocyte line THP-1 (Tsuchiya, S. et al. Int. J. Cancer 26, 171-176, 1980). Although the results are true in THP-1 cells, it is uncertain the results are true in macrophages. The authors should test other macrophages (cell lines) than THP-1 cells such as human HL-60, human MABS-1, human MOBS-1, mouseJ774A.1, mouse RAW264.7, and mouse BMA3.1A7. Alternatively, the authors change the title to “Modulator effect of AT1 receptor knockdown in THP-1 macrophage proinflammatory activity”.

Answer: thanks for the comments, we change the title to “Modulator effect of AT1 receptor knockdown in THP-1 macrophage proinflammatory activity”.

2. “Caledon Laboratories, Canada” (lines 93-94) should be changed to “Caledon Laboratories, Chalk River, Canada”.

Answer: The text was changed

3. “Na2HPO4” (line 96) should be changed to “Na₂HPO₄”.

Answer: The text was changed

4. “KH2PO4” (line 96) should be changed to “KH₂PO₄”.

Answer: The text was changed

5. “Sigma-Aldrich, EE. UU.” (lines 96-97) should be changed to “Sigma-Aldrich, Burlington, MA”.

Answer: The text was changed

6. “25 cm2 flasks” (line 101) should be changed to “25 cm² flasks”.

Answer: The text was changed

7. “5% CO2” (line 102) should be changed to “5% CO₂”.

 Answer: The text was changed

8. “3 x 105 cells” (line 104) should be changed to “3 x 10⁵ cells”.

Answer: The text was changed

9 . “2 x105 cells” (line 108) should be changed to “2 x10⁵ cells”.

Answer: The text was changed

10. “InvivoGen” (line 114) should be changed to “Invivogen”.

Answer: The word InvivoGen could not be change because is the name of the brand

 

11. “2 x105 cells” (line 119) should be changed to “2 x10⁵ cells”.

Answer: The text was changed

12. In Table 1, “NF-KB” should be changed to “NF-κB”.

Answer: The text was changed

13. “R&DSystems, Minneapolis, MN, USA” (lines 145-146) should be changed to “R&DSystems, Minneapolis, MN”.

Answer: The text was changed

14. “Bio-Rad, Calif, USA” (line 148) should be changed to “Bio-Rad, Hercules, CA”.

Answer: The text was changed

15. In Ref. 12, “Wang, C. H., Li, S. H., Weisel, R. D., Fedak, P. W., Dumont, A. S., Szmitko, P., ... & Verma, S.” should be changed to “Wang, C.H., Li, S.H., Weisel, R.D., Fedak, P.W., Dumont, A.S., Szmitko, P., Li, R.K., Mickle, D.A., Verma, S.”.

Answer: The text was changed

16. In Ref. 13, “Recent Patents on Cardiovascular Drug Discovery (Discontinued), 2(1), 23-27” should be changed to “Recent Pat Cardiovasc Drug Discov. 2007;2(1):23-27.”.

Answer: The text was changed

17. In Ref. 28, “Forrester, S. J., Booz, G. W., Sigmund, C. D., Coffman, T. M., Kawai, T., Rizzo, V., ... & Eguchi, S.” should be changed to “Forrester, S.J., Booz, G.W., Sigmund, C.D., Coffman, T.M., Kawai, T., Rizzo, V., Scalia, R., Eguchi, S.”.

Answer: The text was changed

18. In Ref. 39, “Aki, K., Shimizu, A., Masuda, Y., Kuwahara, N., Arai, T., Ishikawa, A., ... & Fukuda, Y.” should be changed to “Aki, K., Shimizu, A., Masuda, Y., Kuwahara, N., Arai, T., Ishikawa, A., Fujita, E., Mii, A., Natori, Y., Fukunaga, Y., Fukuda, Y.”.

Answer: The text was changed

19. In Ref. 40, “Journal of Diabetes Research, 2012” should be changed to “Exp. Diabetes Res. 2012;2012:618923.”.

Answer: The text was changed

20. In Ref. 49, “Valsartan, independientemente del receptor AT1 o PPAR, suprime la activación de macrófagos inducida por LPS y mejora la resistencia a la insulina en adipocitos cocultivados.” should be changed to “Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes.”.

Answer: The text was changed

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I got acquainted with the Acevedo-Villávicencio et al. manuscript submitted for review with interest. The work is carried out step by step, argumentatively, using the necessary experimental evidence of the effect of AT1R gene expression on the production of proinflammatory cytokines (TNF-α, IL-1β), as well as the relationship with blood pressure.

The topic is relevant and has important practical significance, since by medically influencing AT1 receptors (expression of this gene), it is possible to modulate the production of pro- and anti-inflammatory cytokines (IL-1β, TNF-α, IL-10) in macrophages and the expression of the NF-kB gene, and thereby influence blood pressure levels.   The paper reveals the specific mechanisms underlying the association of the AT1 receptor gene (AT1R gene silencing) with cytokine production (IL-1β, TNF-α, IL-10) in macrophages and expression of the NF-kB gene, which is important for the regulation of blood pressure.   The data obtained by the authors will allow us to better understand the mechanisms (involving small interfering RNAs) of the connection between the AT1 receptor and the production of pro- and anti-inflammatory cytokines in the body. Using a human macrophage cell line model, the authors have convincingly shown that the use of telmisartan as an AT1 receptor antagonist (this drug inhibits AT1R activity) leads to a decrease in the synthesis of proinflammatory cytokines such as TNF-α and IL-1β.   At the same time, the authors showed for the first time that   a) telmisartan is less effective than AT1R siRNA as a blocker of angiotensin converting enzyme II in macrophages;   b) AT1R miRNA is a means of modulating inflammatory activity in macrophages by reducing the expression of the NF-kB gene ;   c) AT1R miRNA alters the production of proinflammatory cytokines (IL-1β, TNF-α) and increases the expression of the PPAR-γ gene.   The methodology of the work is adequate to the research tasks set in the work. The experimental research methods used in the work (study of human macrophage cell lines, analysis of small interfering RNAs, isolation of RNA and evaluation of cytokine expression, etc., analysis of cytokine levels, etc.) and statistical analysis are modern and fully allowed the authors to solve all the research tasks. The data obtained by the authors are fully consistent with the currently available ideas about the relationship of the AT1 receptor with cytokine production and its correlation with blood pressure levels.   At the same time, the work experimentally proved that the suppression of AT1R in the human macrophage cell line is an alternative way (!) to modulate the development of inflammation.   The literature sources used in the work allow the authors to fully demonstrate the relevance of their research and discuss the results obtained.   The material presented in the table (the paper presents one table that contains information about the primers used in the work) and figures (the paper presents 5 figures that characterize the expression level of the AT1R, NF-kB, IL-1β, TNF-α, IL-10, PPAR-γ genes, as well as cytokines (proteins) under various experimental conditions (telmisartan, siRNA, control, etc.)) fully demonstrate the working methodology and the results obtained.

There are several suggestions for this work:

1. Eliminate spelling errors (for example, "al-ternative (alternative)", " renin angiotensin aldosterone (renin-angiotensin-aldosterone)").

2. A link to the source should be provided when specifying the GraphPad Prism 10 program used for calculations.

3. It seems important to discuss in the discussion what prospects for clinical use the data obtained by the authors may have.

4. It is recommended in the discussion to outline the prospects for further research on this topic with the potential practical use of the results obtained.

Author Response

Reviewer 2

1. Eliminate spelling errors (for example, "al-ternative (alternative)", " renin angiotensin aldosterone (renin-angiotensin-aldosterone)").

Answer: the mistakes have been changed

2. A link to the source should be provided when specifying the GraphPad Prism 10 program used for calculations.

Answer: The statistical analysis was performed using GraphPad Prism Version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA)

3. It seems important to discuss in the discussion what prospects for clinical use the data obtained by the authors may have.

Answer: The information was added in the discussion (line 386)

4. It is recommended in the discussion to outline the prospects for further research on this topic with the potential practical use of the results obtained.

Answer: The information was added in the discussion (line 386)

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript Modulator effect of AT1 receptor knockdown in macrophage  proinflammatory activity is very excellent work, well presented and well written. The work is of applied nature and results obtained are quite interesting.  The authors have worked on the anti-inflammatory effects of AT1. The results obtained by the authors prove that AT1 receptor antagonist promotes the inhibition of the  secretion of multiple proinflammatory cytokines in macrophages, as well as a decrease in the concentration of reactive oxygen species. The gene expression of IL-1β showed similar results in the group stimulated with Ang II for 1 h. The expression of this cytokine increased  in the group stimulated with Ang II compared with the control group, these findings by the authors are quite interesting. Overall the quality of the manuscript is excellent and deserve for publication. I recommend this manuscript for publication after the incorporation of the following points

I suggest authors to rewrite the abstract to make it more clear and self-explanatory. While going through this interesting manuscript I find some old and ir-relevant references, through the literature i found some interesting and relevant work, i suggest authors to replace:

Replace reference number 1 with 

 

Cao, J., Chen, C., Wang, Y., Chen, X., Chen, Z.,... Luo, X. (2016). Influence of autologous dendritic cells on cytokine‑induced killer cell proliferation, cell phenotype and antitumor activity in vitro. Oncol Lett, 12(3), 2033-2037. doi: 10.3892/ol.2016.4839

Replace reference 5 with 

 

Yuqian Li, R. W. Q. G. (2023). The Roles and Targeting of Tumor-Associated Macrophages. FBL, 28(9), 207. doi: 10.31083/j.fbl2809207

Replace reference 24 with

Liang, W., Liu, H., Zeng, Z., Liang, Z., Xie, H., Li, W.,... Kang, L. (2023). KRT17 Promotes T-lymphocyte Infiltration Through the YTHDF2–CXCL10 Axis in Colorectal Cancer. Cancer Immunology Research, 11(7), 875-894. doi: 10.1158/2326-6066.CIR-22-0814

Replace reference 32 with 

Mao, X., Chen, Y., Lu, X., Jin, S., Jiang, P., Deng, Z., Zhu, X., Cai, Q., Wu, C., & Kang, S. (2023). Tissue resident memory T cells are enriched and dysfunctional in effusion of patients with malignant tumor. Journal of Cancer, 14(7), 1223–1231. https://doi.org/10.7150/jca.83615

Replace reference 43 with 

Wu, R., Xiong, J., Zhou, T., Zhang, Z., Huang, Z., Tian, S.,... Wang, Y. (2023). Quercetin/Anti-PD-1 Antibody Combination Therapy Regulates the Gut Microbiota, Impacts Macrophage Immunity and Reshapes the Hepatocellular Carcinoma Tumor Microenvironment. FBL, 28(12), 327. doi: 10.31083/j.fbl2812327

Incorporation of these interesting work can enhance the potential of the manuscript and eventually beneficial for the readers of the journal

Decision: Minor revision

 

 

 

 

 

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Reviewer 3

I suggest authors to rewrite the abstract to make it more clear and self-explanatory. While going through this interesting manuscript I find some old and irrelevant references, through the literature i found some interesting and relevant work, i suggest authors to replace:

Answer: Thanks for the suggestions we changed the references

Replace reference number 1 with

Cao, J., Chen, C., Wang, Y., Chen, X., Chen, Z.,... Luo, X. (2016). Influence of autologous dendritic cells on cytokine‑induced killer cell proliferation, cell phenotype and antitumor activity in vitro. Oncol Lett, 12(3), 2033-2037. doi: 10.3892/ol.2016.4839

Replace reference 5 with

Yuqian Li, R. W. Q. G. (2023). The Roles and Targeting of Tumor-Associated Macrophages. FBL, 28(9), 207. doi: 10.31083/j.fbl2809207

Replace reference 24 with

Liang, W., Liu, H., Zeng, Z., Liang, Z., Xie, H., Li, W.,... Kang, L. (2023). KRT17 Promotes T-lymphocyte Infiltration Through the YTHDF2–CXCL10 Axis in Colorectal Cancer. Cancer Immunology Research, 11(7), 875-894. doi: 10.1158/2326-6066.CIR-22-0814

Replace reference 32 with

Mao, X., Chen, Y., Lu, X., Jin, S., Jiang, P., Deng, Z., Zhu, X., Cai, Q., Wu, C., & Kang, S. (2023). Tissue resident memory T cells are enriched and dysfunctional in effusion of patients with malignant tumor. Journal of Cancer, 14(7), 1223–1231. https://doi.org/10.7150/jca.83615

Replace reference 43 with

Wu, R., Xiong, J., Zhou, T., Zhang, Z., Huang, Z., Tian, S.,... Wang, Y. (2023). Quercetin/Anti-PD-1 Antibody Combination Therapy Regulates the Gut Microbiota, Impacts Macrophage Immunity and Reshapes the Hepatocellular Carcinoma Tumor Microenvironment. FBL, 28(12), 327. doi: 10.31083/j.fbl2812327

Incorporation of these interesting work can enhance the potential of the manuscript and eventually beneficial for the readers of the journal

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

 

I have carefully reviewed your manuscript and would like to provide some feedback on the content:

 

1. There are two instances of "3.1" in the Results section. Please ensure consistency in numbering the subsections for clarity.

 

2. In paragraph 3.1, it is mentioned "As shown in Figure 2," which should be corrected to "As shown in Figure 1" to align with the correct figure reference.

 

3. It would be beneficial to provide a rationale for choosing the time points of 1 hour and 24 hours for the treatments in the study. Justification for these specific time intervals would enhance the understanding of the experimental design.

 

4. It appears that Figure 3 is missing from the manuscript. Please ensure that all figures are included and labeled correctly for a comprehensive presentation of the data.

 

5. There seems to be an error in the labeling of the groups in the figures. Please review and correct the group labels to accurately represent the experimental conditions.

 

6. It is recommended to provide a detailed explanation of the significance of the Δ symbol in the statistical analysis within the figures. Clarifying the meaning of this symbol will aid readers in interpreting the results accurately.

 

7. Consider expanding the analysis to include the detection of other inflammatory factors to provide a more comprehensive understanding of the impact of AT1 receptor inhibition on inflammation.

      8. To enhance the credibility of the results, consider incorporating additional experiments such as Western blotting (WB) to validate the findings obtained through gene expression analysis.

       Overall, the study shows promise, but addressing these points will strengthen the manuscript and improve its scientific rigor. Thank you for considering these suggestions for the enhancement of your research.

 Best regards,

Comments on the Quality of English Language

Minor editing of English language required according to my editor suggest.

Author Response

Reviewer 4

I have carefully reviewed your manuscript and would like to provide some feedback on the content:

Question

1. There are two instances of "3.1" in the Results section. Please ensure consistency in numbering the subsections for clarity.

Answer: Thank you for your comments, your suggestions has been modified in the manuscript.

2. In paragraph 3.1, it is mentioned "As shown in Figure 2," which should be corrected to "As shown in Figure 1" to align with the correct figure reference.

Answer: thank you for your comment, the text was change in the manuscript.

3. It would be beneficial to provide a rationale for choosing the time points of 1 hour and 24 hours for the treatments in the study. Justification for these specific time intervals would enhance the understanding of the experimental design.

Answer: thank you for your comment. The experimental design at 1 hour of stimulation with Ang II was developed to evaluate the immediate responses in the activation of macrophages, while the evaluation at 24 hours was carried out to analyse the modulation in the release of proinflammatory cytokines. the pathogenesis of cardiovascular diseases (hypertensive cardiac remodeling), the   Inflammation plays an important role in [3] because the Angiotensin (Ang) II, mediates early infiltration of pro-inflammatory cells (monocytes/macrophages) in this affection.

McMaster WG, Kirabo A, Madhur MS, Harrison DG. Inflammation, immunity, and hypertensive end-organ damage. Circ Res. 2015;116:1022–33.

Zhu YC, Zhu YZ, Lu N, Wang MJ, Wang YX, Yao T. Role of angiotensin AT1 and AT2 receptors in cardiac hypertrophy and cardiac remodelling. Clin Exp Pharmacol Physiol. 2003;30:911–8.

O’Rourke SA, Dunne A, Monaghan MG. The role of macrophages in the infarcted myocardium: orchestrators of ECM remodeling. Front Cardiovasc Med. 2019;6:101.

Jia L, Li Y, Xiao C, Du J. Angiotensin II induces inflammation leading to cardiac remodeling. Front Biosci (Landmark Ed) 2012;17:221–31.

4. It appears that Figure 3 is missing from the manuscript. Please ensure that all figures are included and labeled correctly for a comprehensive presentation of the data.

Answer: thank you for your comment, the figure has been added in the manuscript.

5. There seems to be an error in the labeling of the groups in the figures. Please review and correct the group labels to accurately represent the experimental conditions.

Answer: Thank you for your comments: the changes were done in the figures.

 

6. It is recommended to provide a detailed explanation of the significance of the Δ symbol in the statistical analysis within the figures. Clarifying the meaning of this symbol will aid readers in interpreting the results accurately.

Answer: Thank you for your comments: the changes were done in the manuscript.

 

7. Consider expanding the analysis to include the detection of other inflammatory factors to provide a more comprehensive understanding of the impact of AT1 receptor inhibition on inflammation.

Answer: It's an interesting question, we appreciate your comment, our future perspective for future studies is to analyze the signaling cascade of the AT2 receptor, since it could be playing a relevant role in inflammation, because AT1 and AT2 self-regulate.

8. To enhance the credibility of the results, consider incorporating additional experiments such as Western blotting (WB) to validate the findings obtained through gene expression analysis.

Answer: thank you for your comments. We made the decision to measure protein release of macrophages using the ELISA method. We will consider the measurement of intracellular proteins measure out in future research, using the Western blot technique.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Most of points were suitably revised in biology-2987814-v2. A few points to be re-considered/changed (pointed out in <Comments to Authors>) for the benefit of readers. These are given below.

 

<Point>

1.      In Figure 3A & its legend, the authors described, “A) IL-1β gene expression in macrophages stimulated with Ang II (1µM) for 1 h” in legend (lines 280-281). However, the Figure 3A showed “Fold change in mRNA expression TNF-α/GAPDH” (1 h).

2.      In Figure 3B & its legend, the authors described, “B) IL-1β gene expression in macrophages stimulated with Ang II (1µM) for 24 h” (lines 281-282). However, the Figure 3B showed “Fold change in mRNA expression TNF-α/GAPDH” (24 h).

3.      In Figure 3C & its legend, the authors described, “C) TNF-α gene expression in macrophages stimulated with Ang II (1µM) for 1 h” (line 282). However, the Figure 3C showed “Fold change in mRNA expression IL-1β/GAPDH” (1 h).

4.      In Figure 3D & its legend, the authors described, “D) TNF-α gene expression in macrophages stimulated with Ang II (1µM) for 24 h” (lines 282-283). However, the Figure 3D showed “Fold change in mRNA expression IL-1β/GAPDH” (24 h).

5.      In Figure 3E & its legend, “E) IL-10 gene expression in macrophages stimulated with Ang II (1 µM) for 1 h” (lines 283-284). However, there is no Figure 3E.

6.      In Figure 3F & its legend, “F) IL-10 gene expression in macrophages stimulated with Ang II (1µM) for 24 h.” (lines 284-285). However, there is no Figure 3F.

7.      “HyClone, USA” (line 111) should be changed to “HyClone, Logan, UT”.

8.      “The absorbance value (OD 450 nm) was measure” (line 179) should be changed to “The absorbance value (OD 450 nm) was measured”.

9.      In Ref. 5, “FBL, 28(9), 207” should be changed to “Front. Biosci. (Landmark Ed) 2023, 28, 207”.

10.   In Ref. 13, ““Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways,” Cell. Signal., vol. 26, no. 2, pp. 192–197, 2014” should be changed to “Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways. Cell Signal. 2014, 26, 192-197.”.

11.   In Ref. 14, ““Role of renin-angiotensin system in activation of macrophages by modified lipoproteins,” Am. J. Physiol. - Hear. Circ. Physiol., vol. 305, no. 9, pp. 1309–1320, 2013” should be changed to “Role of renin-angiotensin system in activation of macrophages by modified lipoproteins. Am. J. Physiol. Heart Circ. Physiol. 2013, 305, H1309-H320.”.

12.   In Ref. 16, ““Role of angiotensin II type 1 receptor in angiotensin II-induced cytokine production in macrophages,” J. Interf. Cytokine Res., vol. 31, no. 4, pp. 351–361, 2011” should be changed to “Role of angiotensin II type 1 receptor in angiotensin II-induced cytokine production in macrophages. J. Interferon Cytokine Res. 2011, 31, 351-361.”.

13.   In Ref. 23, ““Precise and efficient siRNA design: A key point in competent gene silencing,” Cancer Gene Ther., vol. 23, no. 4, pp. 73–82, 2016” should be changed to “Precise and efficient siRNA design: a key point in competent gene silencing. Cancer Gene Ther. 2016, 23, 73-82.”.

14.   In Ref. 31, “Macrophages and NF-κB in cancer. NF-kB in Health and Disease, 171-184” should be changed to “Macrophages and NF-κB in cancer. Curr. Top. Microbiol. Immunol. 2011, 349, 171-184.”.

15.   In Ref. 33, “La regulación del factor de transcripción NF-κB. Un mediador molecular en el proceso inflamatorio. Revista de investigación clínica, 56(1), 83-92” should be changed to “La regulación del factor de transcripción NF-κB. Un mediador molecular en el proceso inflamatorio [Regulation of NF-κB transcription factor. A molecular mediator in inflammatory process]. Rev. Invest. Clin. 2004, 56, 83-92. Spanish”.

16.   In Ref. 36, “"Proinflammatory actions of angiotensins." Current opinion in nephrology and hypertension 10.3 (2001): 321-329” should be changed to “Proinflammatory actions of angiotensins. Curr. Opin. Nephrol. Hypertens. 2001, 10, 321-329.”.

17.   In Ref. 43, “FBL” should be changed to “Front. Biosci. (Landmark Ed).”.

 

Author Response

Reviewer  1

1. In Figure 3A & its legend, the authors described, “A) IL-1β gene expression in macrophages stimulated with Ang II (1µM) for 1 h” in legend (lines 280-281). However, the Figure 3A showed “Fold change in mRNA expression TNF-α/GAPDH” (1 h).

Answer: thank you for your comment, the text has been modified in legend of the figure 3 A).

2. In Figure 3B & its legend, the authors described, “B) IL-1β gene expression in macrophages stimulated with Ang II (1µM) for 24 h” (lines 281-282). However, the Figure 3B showed “Fold change in mRNA expression TNF-α/GAPDH” (24 h).

Answer: thank you for your observation, the text has been modified in legend of the figure 3 B).

3. In Figure 3C & its legend, the authors described, “C) TNF-α gene expression in macrophages stimulated with Ang II (1µM) for 1 h” (line 282). However, the Figure 3C showed “Fold change in mRNA expression IL-1β/GAPDH” (1 h).

Answer: the text has been modified in legend of the figure 3 C).

4. In Figure 3D & its legend, the authors described, “D) TNF-α gene expression in macrophages stimulated with Ang II (1µM) for 24 h” (lines 282-283). However, the Figure 3D showed “Fold change in mRNA expression IL-1β/GAPDH” (24 h).

Answer: the text has been modified in legend of the figure 3 D).

5. In Figure 3E & its legend,“E) IL-10 gene expression in macrophages stimulated with Ang II (1 µM) for 1 h” (lines 283-284). However, there is no Figure 3E.

Answer: thank you for your comment, the figure has been added in the manuscript.

6. In Figure 3F & its legend,“F) IL-10 gene expression in macrophages stimulated with Ang II (1µM) for 24 h.” (lines 284-285). However, there is no Figure 3F.

Answer: the figure has been added in the manuscript.

7. “HyClone, USA” (line 111) should be changed to “HyClone, Logan, UT”.

Answer: the text has been changed

8. “The absorbance value (OD 450 nm) was measure” (line 179) should be changed to “The absorbance value (OD 450 nm) was measured”.

Answer: the text has been changed

9. In Ref. 5, “FBL, 28(9), 207” should be changed to “Front. Biosci. (Landmark Ed) 2023, 28, 207”.

Answer: the text has been changed

10. In Ref. 13, ““Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways,” Cell. Signal., vol. 26, no. 2, pp. 192–197, 2014” should be changed to “Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways. Cell Signal. 2014, 26, 192-197.”.

Answer: the text has been changed

11. In Ref. 14, ““Role of renin-angiotensin system in activation of macrophages by modified lipoproteins,” Am. J. Physiol. - Hear. Circ. Physiol., vol. 305, no. 9, pp. 1309–1320, 2013” should be changed to “Role of renin-angiotensin system in activation of macrophages by modified lipoproteins. Am. J. Physiol. Heart Circ. Physiol. 2013, 305, H1309-H320.”.

Answer: the text has been changed

12. In Ref. 16, ““Role of angiotensin II type 1 receptor in angiotensin II-induced cytokine production in macrophages,” J. Interf. Cytokine Res., vol. 31, no. 4, pp. 351–361, 2011” should be changed to “Role of angiotensin II type 1 receptor in angiotensin II-induced cytokine production in macrophages. J. Interferon Cytokine Res. 2011, 31, 351-361.”.

Answer: the text has been changed

13. In Ref. 23, ““Precise and efficient siRNA design: A key point in competent gene silencing,” Cancer Gene Ther., vol. 23, no. 4, pp. 73–82, 2016” should be changed to “Precise and efficient siRNA design: a key point in competent gene silencing. Cancer Gene Ther. 2016, 23, 73-82.”.

Answer: the text has been changed

14. In Ref. 31, “Macrophages and NF-κB in cancer. NF-kB in Health and Disease, 171-184” should be changed to “Macrophages and NF-κB in cancer. Curr. Top. Microbiol. Immunol. 2011, 349, 171-184.”.

Answer: the text has been changed

15. In Ref. 33, “La regulación del factor de transcripción NF-κB. Un mediador molecular en el proceso inflamatorio. Revista de investigación clínica, 56(1), 83-92” should be changed to “La regulación del factor de transcripción NF-κB. Un mediador molecular en el proceso inflamatorio [Regulation of NF-κB transcription factor. A molecular mediator in inflammatory process]. Rev. Invest. Clin. 2004, 56, 83-92. Spanish”.

Answer: the text has been changed

16. In Ref. 36, “"Proinflammatory actions of angiotensins." Current opinion in nephrology and hypertension 10.3 (2001): 321-329” should be changed to “Proinflammatory actions of angiotensins. Curr. Opin. Nephrol. Hypertens. 2001, 10, 321-329.”.

Answer: the text has been changed

17. In Ref. 43, “FBL” should be changed to “Front. Biosci. (Landmark Ed).”.

Answer: the text has been changed

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

 

I have carefully reviewed the revised version of your manuscript titled "Effect of AT1 Receptor Gene Silencing on Cytokine Modulation in THP-1 Macrophages" and while I appreciate the efforts made in addressing the previous concerns, there are still some outstanding issues that need to be resolved:

 

1. Figure 3 is still missing from the manuscript. Including this figure is essential for a complete understanding of the results presented in the study.

 

2. In Figure 4, the labeling of the subfigures should be corrected to indicate as A and B for clarity and consistency in the presentation of the data.

 

I kindly request that you address these remaining issues in the manuscript before finalizing it for publication. Once these adjustments are made, the manuscript will be closer to meeting the publication standards.

 

Thank you for your attention to these matters, and I look forward to seeing the revised manuscript with the necessary corrections.

 

Best regards,

Author Response

Reviewer 2

1. Figure 3 is still missing from the manuscript. Including this figure is essential for a complete understanding of the results presented in the study.

 Answer: thank you for your comment, the figure has been added in the manuscript (figure 3 E).

2. In Figure 4, the labeling of the subfigures should be corrected to indicate as A and B for clarity and consistency in the presentation of the data.

Answer: thank you for your comment, the figure 4 has been modified. 

Author Response File: Author Response.docx

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

 I have completed the second round of review for your manuscript titled "Modulatory Effect of AT1 Receptor Inhibition on Proinflammatory Activity of THP-1 Macrophages". I am pleased to inform you that all the issues I previously raised have been successfully addressed in the revised version of the manuscript. I appreciate your attention to detail and the thorough revisions made in response to the feedback provided.

 The clarity of the objectives, the detailed methodology descriptions, and the organization of the results have been significantly improved in the revised manuscript. The changes made have enhanced the overall quality of the study and have effectively addressed the concerns raised during the first round of review.

 Thank you for your hard work and dedication to improving the manuscript. I look forward to seeing your research published.

 

Best regards,